To elucidate molecular mechanisms responsible for the sexually dimorphic phenotype of soluble epoxide hydrolase (sEH) expression, we tested the hypothesis that female-specific down-regulation of sEH expression is driven by estrogen-dependent methylation of the Ephx2 gene. Mesenteric arteries isolated from male, female, ovariectomized female (OV), and OV with estrogen replacement (OVE) mice, as well as the human cell line (HEK293T) were used. Methylation-specific PCR and bisulfite genomic sequencing analysis indicate significant increases in DNA/CG methylation in vessels of female and OVE compared with those of male and OV mice. The same increase in CG methylation was also observed in male vessels incubated with a physiological concentration of 17β-estradiol (17β-E₂) for 48 hours. All vessels that displayed increases in CG methylation were concomitantly associated with decreases in their Ephx2 mRNA and protein, suggesting a methylation-induced gene silencing. Transient transfection assays indicate that the activity of Ephx2 promoter-coding luciferase was significantly attenuated in HEK293T cells treated with 17β-E₂, which was prevented by additional treatment with an estrogen receptor antagonist (ICI). ChIP analysis indicates significantly reduced binding activities of transcription factors (including SP1, AP-1, and NF-κB with their binding elements located in the Ephx2 promoter) in vessels of female mice and human cells treated with 17β-E₂, responses that were prevented by ICI and Decitabine (DNA methyltransferase inhibitor), respectively. In conclusion, estrogen/estrogen receptor-dependent methylation of the promoter of Ephx2 gene silences sEH expression, which is involved in specific transcription factor-directed regulatory pathways.

Roles of soluble epoxide hydrolase (sEH), the enzyme responsible for hydrolysis of epoxyeicosatrienoic acids (EETs) to their diols (DHETs), in the coronary circulation and cardiac function remain unknown. We tested the hypothesis that compromising EET hydrolysis/degradation, via sEH deficiency, lowers the coronary resistance to promote cardiac perfusion and function. Hearts were isolated from wild type (WT), sEH knockout (KO) mice and WT mice chronically treated with t-TUCB (sEH inhibitor), and perfused with constant flow at different pre-loads. Compared to WT controls, sEH-deficient hearts required significantly greater basal coronary flow to maintain the perfusion pressure at 100 mmHg and exhibited a greater reduction in vascular resistance during tension-induced heart work, implying a better coronary perfusion during cardiac performance. Cardiac contractility, characterized by developed tension in response to changes in preload, was potentially increased in sEH-KO hearts, manifested by an enlarged magnitude at each step-wise increase in end-diastolic to peak-systolic tension. 14,15-EEZE (EET antagonist) prevented the adaptation of coronary circulation in sEH null hearts whereas responses in WT hearts were sensitive to the inhibition of NO. Cardiac expression of EET synthases (CYP2J2/2C29) was comparable in both genotypic mice whereas, levels of 14,15-, 11,12- and 8,9-EETs were significantly higher in sEH-KO hearts, accompanied with lower levels of DHETs. In conclusion, the elevation of cardiac EETs, as a function of sEH deficiency, plays key roles in the adaptation of coronary flow and cardiac function.